More Related Content

Recently uploaded(20)


proteoliposomes 👹.pptx

  2. INTRODUCTION •Proteoliposomes are the vesicle systems that mimic lipid membranes ( lipposomes) to which a protein has been incorporated or instered. •During last decade , these systems have gained prominence as tools for biological studies on lipid – protein interactions as well as for their biotecnological Applications.
  3. PRINCIPLE OF FORMATION • The mechanism of the formation of proteoliposomes has long been surprisingly not well defined. • The membrane proteins which are solubilized in Detergent has the ability to integrate into phospholipid bilayers ( Liposomes) when detergent is removed by dilution or dialysis. • The protein, solublized in Detergent is mixed with the prepared Liposomes. The suspension is diluted with the buffer. The protein leaves the detergent and proteins becomes incorporated in the phospholipid bilayer.
  4. PREPARATION OF PROTEOLIPOSOMES STEP BY STEP PROCEDURE The new reconstitution Strategy proceeds in four steps.. , 1. Preparation of large , homogeneous, and unilamellar Liposomes. 2. Addition of detergent to the preformed Liposomes. 3. Addition of Solublized protein. 4. Detergent removal and characterization of the reconstituted products.
  5. LIPOSOMES ; THE PARENT MOLECULE • Liposomes are self assembled closesd spherical structures composed of one or more concentric lipid bilayers and • They range in size from 20 nm to many microns. • They are amphophilic molecules. • They may be unilamellar or multilamellar.
  6. HISTORY OF LIPOSOMES • They are first discover by A.D. Bangham in 1965. • Genesis period ( 1968-75 ): The physical and chemical characteristics of Liposomes have been studied. • Middle age ( 1975 -85): During this period , the advantages, stability and interaction characteristics, physical and chemical properties of liposomes , interactions with cell studied. • Modern era ( after 1985) : Today liposomes are used in different fields , such as biophysics, mathematics, biochemistry, theoretical physics and biology.
  7. METHODS TO PREPARE LIPOSOMES The first stage in the reconstitution strategy is to prepare unilamellar and homogeneous preformed Liposomes. One of the method to achieve this reverse –phase evaporation method. A typical preparation contains 50 mg of phospholipids , usually Solublized in chloroform and dried under high vaccum. Other methods are; Sonication Thin film hydration method followed by extrusion.
  8. ADDITION OF DETERGENT / SOLUBILIZATION • Liposomes prepared are diluted with detergent by adding increasing amount of detergent to aliquoted Liposomes suspension. • Gradual Solubilization of detergent saturated Liposomes Into small lipid – detergent micelles. • At the end of Solubilization process, all Liposomes are Solublized and solution becomes transparent.
  9. ADDITION OF PROTEINS •After the various detergent –phosphilipid mixtures have been equilibrated , the selected membrane protein is added as a monodisperse detergent protein solution. •Protein are incubated for 1 h ( varies according to detergent) with the lipid detergent mixture before detergent removal.
  10. DETERGENT REMOVAL • The last step of reconstitution is related to the removal of detergent from the equilibrated lipid- detergent-ptotein mictures. • Although there are various method of detergent removal , adsorption on Biobeads SM2 has been demonstrated to be the most efficient removal of all kind of detergent. • Washed Biobeads are added directly to each lipid-ptotein – detergent solution. • The solution is incubated with Biobeads several times to remove all of the detergent.
  13. TRANSPORT ACROSS MEMBRANE •Proteoliposomes are widely used to study the function of membrane proteins. •Reconstitution of membrane bound enzymes and transport Proteins with the artificial phospholipid bilayer (liposomes) is one of the most useful techniques to study the functional aspects of these proteins.
  14. ROLE OF LIPOSOMES AS IMMUNOGENS •Liposomes can be prepared mimicking pathogens. •Upon vaccination the immune system primarily recognizes and process them as pathogens. •Liposomes can encapsulate multiple copies of structurally epitopes for a complete set of immune responses. •Proteoliposomes provides an antigenic depot along with the transport of adjuvants which are required for immunity.
  15. ROLE IN NANOTECHNOLOGY • Proteoliposomes are one of the first nanotechnologies to benefit patients with cancer or infectious diseases. • Liposomal anthracyline formulations have demonstrated to have a great impact in oncology. • Deliver highly toxic anti cancer agent in patient with breast carcinoma metastasis. • Long circulating proteoliposomes, coated with polyethylene glycol , can access the angiogenic Vessels , and can effectively deliver the drug as compared to free drug. • Attaching tumor- specific targeting ligands such as monoclonal antibodies to the Liposomes surface results in the accumulation of drug at the tumor site.
  16. TRANSPORTERS OF XENOBIOTICS •Proteoliposomes represent a suitable and up to date tool for studying membrane transporters which physiologically mediate absorption, excretion, trafficking and reabsorption of nutrients and metabolites. •Proteoliposomes have recently been used for studying the interaction of some plasma membrane and mitochondrial transporters with toxic compounds, such as mercurials, H2O2 and some drugs.
  17. LIMITATIONS 1.Liposomes delivered intervaenously can destroyed by mononuclear phagocyte system. 2.Interaction between Liposomes and lipoproteins in the blood, which may cause the Liposomes to leak. 3.Proteoliposomes Reconstitution by mechanical means drastically limited because of local probe heating